The HIV-1 Gag protein orchestrates co-factor packaging, assembly and release of the immature virion. As the virion emerges, Gag is then processed by the viral protease to produce the mature CA protein, which reassembles into the conical capsid that organizes the viral core for uncoating and replication in a new host cell. Research during the previous funding period has revealed the structure of the viral capsid and begun to define its roles in viral replication. We now propose to expand our studies of particle assembly to include: 1) structural studies of CA proteins aimed at defining the molecular details of CA-ligand and CA-CA interactions, 2) structural studies of larger Gag protein fragments aimed at understanding how Gag organizes the immature capsid and changes conformation during viral assembly, and 3) development of assays that will be used to study viral capsid functions in vitro. Preliminary results include the growth of high quality crystals of a protein spanning the first 278 residues of HIV-1 Gag, the demonstration that the amino- terminal end of CA can adopt at least three distinct conformations, and the implementation of procedures for viral core preparation and endogenous reverse transcription. We will also study how the assembling Gag lattice interacts with cytoskeletal proteins during viral assembly. Although roles for cytoskeletal proteins in HIV-1 assembly have not been characterized previously, we have identified a series of protein-protein interactions that link HIV-1 Gag and its cellular binding partner, TSG101, to proteins that normally control actomyosin organization and activation. Both overexpression and inhibition of these regulatory proteins block virus assembly and release without altering Gag expression levels, indicating that they play a direct role in particle assembly, possibly by helping to remodel the cortical actin cytoskeleton and/or to drive particle envelopment. Proposed studies will define this protein interaction network in detail, and define its role in HIV-1 particle assembly.